Screening of compounds for BGT1 transporter activity

ABSTRACT

BGT1 is consistently expressed at high levels in brain microvessel endothelial cells. Disclosed herein are assays for determining whether a test material/molecule is a substrate for, and/or is actively transported by, the BGT1 transporter, and therefore a candidate substrate for crossing the blood brain barrier. The assays are useful in screening for therapeutic, cytotoxic or imaging compounds used in the treatment or diagnosis of neurological diseases.

This application claims benefit of U.S. Provisional ApplicationNo.60/703,710 filed Jul. 29, 2005, which is incorporated by referenceherein in its entirety.

TECHNICAL FIELD

The disclosures herein relate to assays and methods of using the samefor screening compounds and/or chemical moieties for their ability to beactively transported across the blood brain barrier.

BACKGROUND

The capillaries that supply blood to the tissues of the brain constitutethe blood brain barrier (Goldstein et al. (1986) Scientific American255:74-83; Pardridge, W. M. (1986) Endocrin. Rev. 7:314-330). Theendothelial cells, which form the brain capillaries, are different fromthose found in other tissues in the body. Brain capillary endothelialcells are joined together by tight intercellular junctions, which form acontinuous wall against the passive diffusion of molecules from theblood to the brain and other parts of the central nervous system (CNS).These cells are also different in that they have few pinocytic vesicles,which in other tissues allow somewhat unselective transport across thecapillary wall. Also lacking are continuous gaps or channels runningbetween the cells that would allow unrestricted passage.

The blood-brain barrier functions to ensure that the environment of thebrain is constantly controlled. The levels of various substances in theblood, such as hormones, amino acids and ions, undergo frequent smallfluctuations, which can be brought about by activities such as eatingand exercise (Goldstein et al., cited supra). If the blood brain barrierdid not protect the brain from these variations in serum composition,the result could be uncontrolled neural activity.

The isolation of the brain from the bloodstream is not complete. If thiswere the case, the brain would be unable to function properly due to alack of nutrients and because of the need to exchange chemicals with therest of the body. The presence of specific transport systems within thecapillary endothelial cells assures that the brain receives, in acontrolled manner, all of the compounds required for normal growth andfunction. In many instances, these transport systems consist ofmembrane-associated proteins, which selectively bind and transportcertain molecules across the barrier membranes. These transporterproteins are known as solute carrier transporters.

The problem posed by the blood brain barrier is that, in the process ofprotecting the brain, it excludes many potentially useful therapeuticagents. Presently, only substances that are sufficiently lipophilic canpenetrate the blood-brain barrier (Goldstein et al., cited supra;Pardridge, W. M., cited supra). Some drugs can be modified to make themmore lipophilic and thereby increase their ability to cross the bloodbrain barrier. However, each modification must be tested individually oneach drug and the modification can alter the activity of the drug.

Because the blood brain barrier is composed of brain microvesselendothelial cells, these cells have been isolated and cultured for usein in vitro model systems for studying the blood brain barrier (Bowmanet al., Brain microvessel endothelial cells in tissue culture: A modelfor study of blood-brain barrier permeability, Ann. Neurol. 14, 396-402(1983); Audus and Borchardt, Characterization of an in vitro blood-brainbarrier model system for studying drug transport and metabolism, Pharm,Res. 3, 81-87 (1986)). In vitro model systems of the blood brain barrierhave been successfully derived from bovine, canine, human, murine,porcine, and rat cells, and have similar permeability properties due tosimilarity of the physiological characteristics of the blood brainbarrier among mammals (Cserr et al., Blood-brain interfaces invertebrates: a comparative approach, Am. J. Physiol. 246, R277-R288(1984); Audus et al., The use of cultured epithelial and endothelialcells for drug transport and metabolism studies, Pharm. Res. 7, 435-451(1990)). In these models, the cultured endothelial cells retain thecharacteristics of brain endothelial cells in vivo, such as morphology,specific blood brain barrier enzyme markers, and tight intercellularjunctions. The cells can also be used for the study of passivediffusion, carrier mediated transport, and metabolism to specificfactors affecting the blood brain barrier permeability. However,passaging of brain microvessel endothelial cells results in loss ofspecific endothelial and blood brain barrier markers as well as tightintercellular junctions (Brightman and Neuwelt (ed.), Implications ofthe blood-brain barrier and its manipulation, Vol. 1, Plenum Medical,New York, pp.53-83 (1989)).

Currently, primary cultures of brain microvessel endothelial cells arethe principal tool for in vitro prediction of blood brain barrierpermeability. Isolated and cultured primary brain cells developedpreviously have exhibited different properties primarily due toconsiderable variability in the starting material. For example, withrespect to transcellular transport, rigorous comparison of data betweendifferent laboratories has been very difficult (Pardridge et al.,Comparison of in vitro and in vivo models of drug transcytosis throughthe blood-brain barrier, J. Pharmacol. Exp. Thera. 253, 884-891 (1990);Masereeuw et al., In vitro and in vivo transport of zidovudine (AZT)across the blood-brain barrier and the effect of transport inhibitors,Pharm. Res., 11, 324-330 (1994)). Passaging primary cells can affect thedifferentiation of cells and lead to the selection of the most rapidlyproliferating clones. Furthermore, the expression of some marker enzymessuch as gamma-glutamyl transpeptidase as well as tight junctionalcomplexity has been shown to decrease with time in culture and passagenumber (Meresse et. al., Bovine brain endothelial cells express tightjunctions and monoamine oxidase activity in long-term culture, J.Neuorchem. 53, 1363-1371 (1989)). Some transporter substrates have beendemonstrated to accumulate in the brain (see U.S. Pat. No. 6,489,302).

Thus, it is apparent that the presently available clones of immortalizedbrain microvessel endothelial cell cultures suffer from individualdrawbacks in terms of phenotype expression and homogeneic maintenance ofthat expression. This leads to difficulties with respect to accuracy andreproducibility in studies utilizing brain microvessel endothelial cellsto model passage of chemical compounds and moieties, e.g., potentialtherapeutic compounds and/or drug moieties, across the blood brainbarrier.

SUMMARY

Disclosed herein are methods of screening agents, conjugates orconjugate moieties for the ability to enter the CNS by crossing theblood brain barrier in order to treat or diagnose conditions within theCNS. These methods entail providing a cell expressing the BGT1transporter, the transporter being situated in the plasma membrane ofthe cell. The cell is contacted with an agent, conjugate, or conjugatemoiety. Whether the agent, conjugate, or conjugate moiety passes throughthe plasma membrane via the BGT1 transporter is determined. If themethod comprises contacting the cell with an agent, the agent is aneuropharmaceutical agent or an imaging component. If the methodcomprises contacting the cell with a conjugate, the conjugate comprisesan agent that is a neuropharmaceutical agent or an imaging component. Ifthe method comprises contacting the cells with a conjugate moiety, themethod further comprises linking the conjugate moiety to an agent thatis a neuropharmaceutical agent or an imaging component.

In some methods, the cell endogenously expresses the BGT1 transporter.In other methods a nucleic acid molecule encoding the BGT1 transporterhas been transfected or injected into the cell. In some methods the cellis a brain microvessel endothelial cell. In other methods the cell is anoocyte. In other methods the cell is a human embryonic kidney (HEK)cell. In other methods the cell is a Madin Darby canine kidney (MDCK)cell. In other methods the cell is a porcine kidney epithelial (LLCPK)cell. In other methods the cell is a Chinese hamster ovary (CHO) cell.In still other methods, the cell is constructed to conditionally expressthe transporter.

In some methods the agent, conjugate, or conjugate moiety comprises anamino acid. In some methods the agent, conjugate, or conjugate moiety isadministered to an undiseased animal and any toxic effects aredetermined. In some methods the neuropharmaceutical agent is a cytotoxicneuropharmaceutical agent selected from the group consisting ofplatinum, nitrosourea, a phosphoramide group that is selectivelycytotoxic to brain tumor cells, nitroimidazole, and nitrogen mustard.

Disclosed herein are methods of screening agents, conjugates orconjugate moieties for the ability to enter the CNS by crossing theblood brain barrier wherein a cell used for testing is a brainmicrovessel endothelial cell that is one of a plurality of brainmicrovessel endothelial cells forming a polarized monolayer. An agent,conjugate, or conjugate moiety is contacted to one side of the polarizedmonolayer and whether the agent, conjugate, or conjugate moiety istransported into the brain microvessel endothelial cells or to theopposite side of the polarized monolayer is determined. Some methodsfurther comprise administering the agent, conjugate, or conjugate moietyto a peripheral tissue of an animal and measuring the amount of agent,conjugate, or conjugate moiety that passes through the blood brainbarrier into the brain of the animal.

Disclosed herein are methods of screening an agent, conjugate, orconjugate moiety for neuropharmacological activity useful for treatingneurological disorders. In these methods, one determines whether theagent, conjugate, or conjugate moiety is transported through the BGT1transporter. One then administers the agent, conjugate, or conjugatemoiety to a test animal and determines whether the agent, conjugate, orconjugate moiety is actively transported across the blood brain barrierby measuring agent, conjugate, or conjugate moiety concentrations foundin the CNS of the animal. For those agents, conjugates or conjugatemoieties that are transported in sufficient quantities, the agents,conjugates or conjugate moieties can be further tested in animalssuffering from a particular neurological disorder to determine whetherthe agents, conjugates or conjugate moieties have the requisitetherapeutic neuropharmacological activity for treating such neurologicaldisorder.

Also disclosed herein are methods for in vitro screening of agents,conjugates or conjugate moieties for improved retention in the CNS. Inthese methods, one determines the substrate properties of a compound onboth uptake transporters and efflux transporters. An agent, conjugate,or conjugate moiety is first tested for activity on the BGT1transporter. The agent, conjugate, or conjugate moiety is then testedfor substrate activity on an efflux transporter, such as P Glycoprotein(PgP). Those agents, conjugates or conjugate moieties active on both theefflux transporter and BGT1 are then modified and tested for a reductionof efflux substrate activity and retested for retention of activity onthe BGT1 transporter. This iterative process produces an agent,conjugate, or conjugate moiety with an increased ratio of substrateactivities in the uptake and efflux systems, and improved retention ofpharmacological levels of the modified agent, conjugate, or conjugatemoiety in the CNS.

Disclosed herein are methods of screening an agent, conjugate, orconjugate moiety for capacity to be transported into the brain,comprising determining whether the agent, conjugate, or conjugate moietyspecifically binds to the BGT1 transporter, contacting the agent to oneside of a polarized monolayer of cells, and determining whether theagent is actively transported across the polarized monolayer. In somemethods the specific binding is determined by contacting a cellexpressing the BGT1 transporter, the transporter being situated in theplasma membrane of the cell, with a substrate of the BGT1 transporter,and determining whether the agent inhibits transport of the substrateacross the polarized monolayer.

Disclosed herein are pharmaceutical compositions comprising atherapeutic neuropharmaceutical agent, a cytotoxic neuropharmaceuticalagent or an imaging component linked to a conjugate moiety to form aconjugate in which the conjugate moiety has a higher V_(max) for theBGT1 transporter than the therapeutic neuropharmaceutical agent,cytotoxic neuropharmaceutical agent or imaging component alone. Somepharmaceutical compositions have at least 5 times the V_(max) for BGT1than the neuropharmaceutical agent or the imaging component alone. Insome pharmaceutical compositions the conjugate has a V_(max) for BGT1that is at least 5% of the V_(max) for BGT1 of a compound selected fromthe group comprising betaine and GABA (γ-aminobutyric acid). In somepharmaceutical compositions the conjugate has a lower V_(max) for anefflux transporter than the neuropharmaceutical agent or the imagingcomponent alone.

Disclosed herein are methods of formulating a therapeuticneuropharmaceutical agent, a cytotoxic neuropharmaceutical agent or animaging component. These methods entail linking the therapeuticneuropharmaceutical agent, the cytotoxic neuropharmaceutical agent orthe imaging component to a conjugate moiety to form a conjugate, whereinthe conjugate moiety has a greater V_(max) for the BGT1 transporter thanthe therapeutic neuropharmaceutical agent, the cytotoxicneuropharmaceutical agent or the imaging component alone. The conjugateis formulated with a pharmaceutical carrier as a pharmaceuticalcomposition.

Disclosed herein are methods of delivering a therapeuticneuropharmaceutical agent, a cytotoxic neuropharmaceutical agent or animaging component. The methods involve administering to a patient apharmaceutical composition comprising a therapeutic neuropharmaceuticalagent, a cytotoxic neuropharmaceutical agent or an imaging componentlinked to a conjugate moiety to form a conjugate, wherein the conjugatehas a higher V_(max) for the BGT1 transporter than the therapeuticneuropharmaceutical agent, cytotoxic neuropharmaceutical agent orimaging component alone, whereby the conjugate passes through brainmicrovessel endothelial cells which make up the blood brain barrier, viathe BGT1 transporter, into the CNS of the patient. Also disclosed hereinare methods of delivering a conjugate, comprising administering to apatient a pharmaceutical composition comprising a neuropharmaceuticalagent or imaging component linked to a conjugate moiety to form theconjugate, wherein the conjugate has a higher V_(max) for the BGT1transporter than the neuropharmaceutical agent or imaging componentalone. In some methods the V_(max) of the conjugate is at least two-foldhigher than that of the neuropharmaceutical agent or imaging componentalone. In some methods the neuropharmaceutical agent is a cytotoxicneuropharmaceutical selected from the group consisting of platinum,nitrosourea, a phosphoramide group selectively cytotoxic to brain tumorcells, nitroimidazole, and nitrogen mustard.

Disclosed herein are methods of treating neurological disorders. Thesemethods entail administering to a patient an effective amount of anagent that is transported by BGT1, wherein the agent is a conjugatecomprising a therapeutic neuropharmaceutical agent, a cytotoxicneuropharmaceutical agent or an imaging component linked to a conjugatemoiety.

Disclosed herein are methods of screening an agent for decreased sideeffects in the central nervous system (CNS), comprising providing anagent having a pharmacological activity, wherein the pharmacologicalactivity is useful for treating a disease present in a tissue other thanthe CNS, and the pharmacological activity results in undesired sideeffects in the CNS if the agent enters the CNS, modifying the agent,providing a cell expressing at least one efflux transporter protein thattransports substrates out of the CNS, contacting the cell with themodified agent, and determining whether the modified agent istransported by the at least one efflux transporter protein with a higherV_(max) than the agent, a higher V_(max) indicating that themodification increases the capacity of the modified agent relative tothe agent to be transported out of the CNS, thereby decreasing undesiredside effects in the CNS.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structures of known substrates of the BGT1 transporter.

FIG. 2 shows competition assays with HEK-TREx-BGT1 cells using ³H-GABAas a substrate, and GABA as a competitor.

FIG. 3 shows a direct uptake assay with HEK-TREx-BGT1 cells using GABAas a substrate. The specific uptake of GABA into cells induced toexpress hBGT1 is shown. Specific uptake was determined by subtractingthe values obtained in cells not induced to express HBGT1 from those inthe induced cells and graphed vs. the test concentration of eachsubstrate.

DEFINITIONS

“Transport by passive diffusion” refers to transport of an agent that isnot mediated by a specific transporter protein. An agent that issubstantially incapable of passive diffusion has a permeability across astandard cell monolayer (e.g., Caco-2 or MDCK cells or an artificialbilayer (PAMPA)) of less than 5×10⁻⁶ cm/sec, and usually less than1×10⁻⁶ cm/sec in the absence of an efflux mechanism.

A “substrate” of a transporter protein is a compound whose uptake intoor passage through the plasma membrane of a cell is facilitated at leastin part by a transporter protein.

The term “ligand” of a transporter protein includes compounds that bindto the transporter protein. Some ligands are transported and are therebyalso substrates. Some ligands inhibit or antagonize transport of asubstrate by the transporter protein. Some ligands bind in a mannernon-competitive with substrates and modulate the transport of substratesby the transporter protein.

The term “neuropharmaceutical agent” is used to describe a compound thathas or may have a pharmacological activity in the treatment orprophylaxis of a neurological disorder. Neuropharmaceutical agentsinclude compounds that are known drugs, compounds for whichpharmacological activity has been identified but which are undergoingfurther therapeutic evaluation, and compounds that are members ofcollections and libraries that are to be screened for a pharmacologicalactivity. The neuropharmaceutical agent can be a compound having atherapeutic, prophylactic or cytotoxic effect on a neurological diseaseincluding any condition that affects biological functioning of thecentral nervous system. Examples of neurological diseases include cancer(e.g., brain tumors), Acquired Immune Deficiency Syndrome (AIDS),stroke, epilepsy, Parkinson's disease, multiple sclerosis,neurodegenerative disease, trauma, depression, Alzheimer's disease,migraine, pain, or a seizure disorder. Classes of neuropharmaceuticalagents include proteins, antibiotics, adrenergic agents,anticonvulsants, small molecules, nucleotide analogs, chemotherapeuticagents, anti-trauma agents, peptides and other classes of agents used intreatment or prophylaxis of a neurological disorder. Examples of suchproteins include CD4 (including soluble portions thereof), growthfactors (e.g., nerve growth factor and interferon), dopaminedecarboxylase and tricosanthin. Examples of such antibiotics includeamphotericin B, gentamycin sulfate, and pyrimethamine. Examples of suchadrenergic agents (including blockers) include dopamine and atenolol.Examples of such chemotherapeutic agents include adriamycin,methotrexate, cyclophosphamide, etoposide, and carboplatin. An exampleof an anticonvulsant that can be used is valproate and an anti-traumaagent that can be used is superoxide dismutase. Examples of suchpeptides are somatostatin analogues and enkephalinase inhibitors.Nucleotide analogs that can be used include azido thymidine (hereinafterAZT), dideoxy Inosine (ddI) and dideodxy cytodine (ddc).

The term “agent” is used to describe a compound that has or may have apharmacological activity. Agents include compounds that are known drugs,compounds for which pharmacological activity has been identified butwhich are undergoing further therapeutic evaluation, and compounds thatare members of collections and libraries that are to be screened for apharmacological activity.

A “pharmacological” activity means that an agent exhibits an activity ina screening system that indicates that the agent is or may be useful inthe prophylaxis or treatment of a disease. The screening system can bein vitro, cellular, animal or human. Agents can be described as havingpharmacological activity notwithstanding that further testing may berequired to establish actual prophylactic or therapeutic utility intreatment of a disease.

An agent is “orally active” if it can exert a pharmacological activitywhen administered via an oral route.

A “peripheral tissue” means a tissue other than the CNS.

A “conjugate” refers to a compound comprising a neuropharmaceuticalagent or imaging component and a chemical moiety bound thereto, whichmoiety by itself or in combination with the neuropharmaceutical agent orimaging component renders the conjugate a substrate for activetransport, for example rendering the conjugate to be a substrate for atransporter protein. The chemical moiety may or may not be subject tocleavage from the neuropharmaceutical agent or imaging component uponuptake and metabolism of the conjugate in the patient's body. In otherwords, the moiety may be cleavably bound to the neuropharmaceuticalagent or imaging component or non-cleavably bound to theneuropharmaceutical agent or imaging component. The bond can be a direct(i.e., covalent) bond or the bond can be through a linker. In caseswhere the bond/linker is cleavable by metabolic processes, theneuropharmaceutical agent or imaging component, or a further metaboliteof the neuropharmaceutical agent or imaging component, is thetherapeutic or imaging entity. In cases where the bond/linker is notcleavable by metabolic processes, the conjugate itself is thetherapeutic or imaging entity. Most typically, the conjugate comprises aprodrug having a metabolically cleavable moiety, where the conjugateitself does not have pharmacological activity but the component to whichthe moiety is cleavably bound does have pharmacological activity.Typically, the moiety facilitates therapeutic use of theneuropharmaceutical agent or imaging component by promoting uptake ofthe conjugate via a transporter. Thus, for example, a conjugatecomprising a neuropharmaceutical agent and a conjugate moiety may have aV_(max) for a transporter that is at least 2, 5, 10, 20, 50, or 100-foldhigher than that of the neuropharmaceutical agent or imaging componentalone. A conjugate moiety can itself be a substrate for a transporter orcan become a substrate when linked to the neuropharmaceutical agent orimaging component. Examples of preferred conjugate moieties are betaineand GABA. Thus, a conjugate formed from a neuropharmaceutical agent orimaging component and a conjugate moiety can have higher CNS uptakeactivity than the neuropharmaceutical agent, the imaging component, orthe conjugate moiety alone.

A “neuropharmacological” activity means that a neuropharmaceutical agentexhibits an activity in a screening system that indicates that theneuropharmaceutical agent is or may be useful in the prophylaxis ortreatment of a neurological disease. The screening system can be invitro, cellular, animal or human. Neuropharmaceutical agents can bedescribed as having neuropharmacological activity notwithstanding thatfurther testing may be required to establish actual prophylactic ortherapeutic utility in treatment of a disease.

V_(max) and K_(m) of a compound for a transporter are defined inaccordance with convention. V_(max) is the number of molecules ofcompound transported per second at saturating concentration of thecompound. K_(m) is the concentration of the compound at which thecompound is transported at half of V_(max). When the goal is totransport an agent, conjugate, or conjugate moiety into the CNS, a highV_(max) for an influx transporter such as BGT1 is generally desirable.Likewise for the same goal, a low value of K_(m) is typically desirablefor transport of a compound present at low blood concentrations. In somecases a high value of K_(m) is acceptable for the transport of compoundspresent at high concentrations in the blood. For these reasons, theintrinsic capacity of a compound to be transported by a particulartransporter is usually expressed as the ratio V_(max) of thecompound/V_(max) of a reference compound known to be a substrate for thetransporter. V_(max) is affected both by the intrinsic turnover rate ofa transporter (molecules/transporter protein) and transporter density inthe plasma membrane, which depends on expression level. In certaininstances, the goal is to avoid transport into the CNS. In theseinstances, a low V_(max) for all influx transporters and a high V_(max)for all efflux transporters expressed in the blood brain barrier aredesirable.

“EC50,” or “effective concentration 50,” is a measurement of thesubstrate concentration that results in a turnover rate 50% of themaximal turnover rate for the substrate (0.5 V_(max)).

A plasma membrane containing a monolayer of cells in physical contactwith each other and having different sets of proteins embedded in theplasma membranes facing either side of the monolayer is described asbeing “polarized”. For example, brain microvessel endothelial cells inthe blood brain barrier have a luminal side facing capillaries andexposed to blood, and an abluminal side facing cells of the centralnervous system and exposed to cerebrospinal fluid. The luminal plasmamembrane contains a different set of transmembrane andmembrane-associated components than the abluminal plasma membrane of thesame cell. Brain microvessel endothelial cells in culture can also bepolarized, where the cells form a monolayer in culture that has aluminal and abluminal side. MDCK cells, when grown on filter membranesin transwell dishes, form a polarized monolayer in which one side of themonolayer is the apical side and the other is the basolateral side.

“Sustained release” refers to release of a therapeutic or prophylacticamount of a drug or an active metabolite thereof over a period of timethat is longer than a conventional formulation of the drug. For oralformulations, the term “sustained release” typically means release ofthe drug within the gastrointestinal tract lumen over a period of fromabout 2 to about 30 hours, more typically over a period of about 4 toabout 24 hours. Sustained release formulations achieve therapeuticallyeffective concentrations of the drug in the systemic blood circulationover a prolonged period of time relative to that achieved by oraladministration of a conventional formulation of the drug. “Delayedrelease” refers to release of the drug or an active metabolite thereofinto the gastrointestinal lumen after a delay time period, typically adelay of about 1 to about 12 hours, relative to that achieved by oraladministration of a conventional formulation of the drug.

The phrase “specifically binds” when referring to a substrate or ligandof the BGT1 transporter refers to a specific interaction between asubstrate or ligand and the BGT1 transporter in which the substrate orligand binds preferentially with the BGT1 transporter and does not bindin a significant amount to most or any other proteins present in abiological sample. A substrate or ligand that specifically binds to theBGT1 transporter often has an association constant of 10-10³ M⁻¹, 10⁵M⁻¹, 10⁶ M⁻¹, or 10⁷ M⁻¹, preferably 10⁸ M⁻¹ to 10⁹ M⁻¹ or higher.However, some substrates or ligands of BGT1 transporters have much loweraffinities and yet the binding can still be shown to be specific.Substrates of BGT1 can specifically bind to BGT1 and other proteins suchas efflux transporters without specifically binding to other proteins.

“P_(app),” or “apparent permeability,” is a value that reflects thepermeability of a test compound through a cell layer such as a polarizedmonolayer. The equation for determining P_(app) is as follows:

$P_{app} = \frac{{V \cdot {dC}}\mspace{14mu}\left( {{cm}\text{/}\sec} \right)}{A \cdot C_{0} \cdot {dt}}$

where,

-   -   V=volume of receiving chamber (in cm³, i.e., ml);    -   dC/dt=steady state rate of appearance of applied compound in        receiving chamber after primary lag time (in μM/sec);    -   C₀=concentration of compound in the donor chamber (in μM); and    -   A=area of the cell layer (in cm²).

“Allelic variants” at the DNA level are the result of genetic variationbetween individuals of the same species. Some allelic variants at theDNA level that cause substitution, deletion or insertion of amino acidsin proteins encoded by the DNA result in corresponding allelic variationat the protein level.

“Cognate forms” of a gene refers to variation between structurally andfunctionally related genes between species. For example, the human geneshowing the greatest sequence identity and closest functionalrelationship to a mouse gene is the human cognate form of the mousegene.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generallyAusubel et al., supra).

Another example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information web site. This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra.).These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always>0) and N (penalty scorefor mismatching residues; always<0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. For identifying whether a nucleic acid orpolypeptide is within the scope hereof, the default parameters of theBLAST programs are suitable. The BLASTN program (for nucleotidesequences) uses as defaults a word length (W) of 11, an expectation (E)of 10, M=5, N=−4, and a comparison of both strands. For amino acidsequences, the BLASTP program uses as defaults a word length (W) of 3,an expectation (E) of 10, and the BLOSUM62 scoring matrix. The TBLASTNprogram (using protein sequence for nucleotide sequence) uses asdefaults a word length (W) of 3, an expectation (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA89:10915 (1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

DETAILED DESCRIPTION

I. General

BGT1 is shown herein to be expressed at high levels in brain microvesselendothelial cells. This finding can be used to generate or isolateconjugates and agents having neuropharmacological or imaging activityuseful for treatment, prophylaxis or diagnosis of neurological diseases.The invention provides methods of identifying agents, conjugates orconjugate moieties that are substrates for BGT1. For therapeuticpurposes, agents or conjugates having inherent neuropharmacologicactivity can be screened to determine whether they are substrates forBGT1. Alternatively, a conjugate moiety lacking such activity can bescreened, and linked to a neuropharmacologic agent after screening.Agents or conjugates that have both neuropharmacologic activity and aresubstrates for BGT1 are preferentially transported into the CNS via BGT1transporters after administration to a patient. Such an agent orconjugate by itself or in combination with another agent is effective intreatment or prophylaxis of a neurological disease. An analogousapproach is used for imaging features of the brain. Agents andconjugates that have an imaging component and are substrates for BGT1are preferentially transported into the CNS via BGT1 transporters. Theimaging component is then detected by various methods such as detectingradioactive decay of the imaging component. The agents and conjugatescan be used to image brain tumors overexpressing the BGT1 transporter.Optionally, the agents or conjugates have inherent affinity for, or areprovided with a conjugate moiety that confers affinity for, a particularantigen or cell type within the brain. For example, the agents orconjugates can be provided with a targeting moiety to Aβ to allowimaging of plaques in Alzheimer's patients.

II. BGT1 Transporter

The family of sodium and chloride coupled neurotransmitter transporterscontains at least 16 members in humans (SLC6A1-16). Neurotransmittertransporters have 10-13 putative transmembrane domains, with both theamino and carboxy termini located on the cytoplasmic side. Mostneurotransmitter transporters only transport neurotransmitters and aminoacids. One member of this family is BGT1 (SLC6A12), which mediates thecellular uptake of betaine, beta-alanine, and gamma-aminobutyric acid(GABA). BGT1 transport is dependent on the co-transport of sodium andchloride ions.

It is now shown that BGT1 is highly expressed in brain microvesselendothelial cells. It is desirable to generate agents, conjugates, andconjugate moieties for transport into the CNS that have activity forBGT1 due to this high expression level. The GenBank accession number forhuman BGT1 is NM_(—)003044 (SEQ ID NO: 1). Unless otherwise apparentfrom the context, reference to a transporter includes the amino acidsequence described in or encoded by the GenBank reference numberNM_(—)003044, and, allelic, cognate and induced variants and fragmentsthereof retaining essentially the same transporter activity. Usuallysuch variants show at least 90% sequence identity to the exemplaryGenbank nucleic acid or amino acid sequence.

III. Methods of Screening to Identify BGT1 Substrates

Agents known or suspected to have a neuropharmaceutical activity or tocomprise an imaging component can be screened directly for theircapacity to act as substrates of BGT1. Alternatively, conjugate moietiescan be screened as substrates, and the conjugate moieties are thenlinked to a neuropharmaceutical agent or imaging component. In suchmethods, the conjugate moieties can optionally be linked to aneuropharmaceutical agent or imaging component, or other molecule duringthe screening process. If another molecule is used in place of aneuropharmaceutical agent or imaging component, the molecule can bechosen to resemble the structure of a neuropharmaceutical agent orimaging component ultimately intended to be linked to the conjugatemoiety for neuropharmaceutical use. Alternatively, a conjugate moietycan be screened for a substrate activity alone and linked to aneuropharmaceutical agent or imaging component after screening.

Preferred substrates for BGT1 are amino acids such as betaine,beta-alanine, and GABA (see, e.g., Deves and Boyd, 1998, Transportersfor Cationic Amino Acids in Animal Cells: Discovery, Structure, andFunction, Physiological Reviews, 78:2, 847-545). The structure of eachcompound is depicted in FIG. 1.

Betaine, beta-alanine, and GABA are examples of BGT1 substrates that arecandidates for conjugation to therapeutic neuropharmaceutical agents,cytotoxic neuropharmaceutical agents and imaging components.

In some screening methods, the cells are transfected with DNA encodingthe BGTl transporter. LLCPK (porcine kidney epithelial), HEK (humanembryonic kidney), and CHO (Chinese hamster ovary) cells, for example,are suitable for transfection. Oocytes can be injected with BGT1 cRNA toexpress the BGT1 transporter. In some methods, the only transporterexpressed by the cells is the BGT1 transporter. In other methods, cellsexpress BGT1 in combination with other transporters. In still othermethods, agents, conjugate moieties, or conjugates are screened ondifferent cells expressing different transporters. Agents, conjugatemoieties, or conjugates can be screened either for specificity for theBGT1 transporter or for transport into cells endogenously expressing aplurality of transporters. In some methods, the results of a screeningmethod (e.g., a competition uptake, exchange or direct uptake assay)using a cell expressing the BGT1 transporter can be compared with theresults of a control cell(s) lacking the BGT1 transporter or in thepresence of a specific inhibitor of the BGT1 transporter.

In some methods, cells endogenously expressing the BGT1 transporter areused. Brain microvessel endothelial cells, for example, endogenouslyexpress the BGT1 transporter, as demonstrated in Example 1. Agents,conjugate moieties, or conjugates can be screened for transport intocultured brain microvessel endothelial cells. Passaging cultures ofbrain microvessel endothelial cells typically causes the cells to losedifferentiation characteristics such as the ability to form tightjunctions. The propensity of passaged cells to lose differentiationcharacteristics can be avoided through the use of brain microvesselendothelial cells that are transformed with an SV40 large T antigen (seeTerasaki et al., Drug Discovery Today 8:944-954 (2003)). Inducibleexpression of the SV40 large T antigen allows cells to divide when theantigen is expressed and differentiate when the antigen is notexpressed. Brain microvessel endothelial cells can be isolated fromanimals transgenic for the SV40 large T antigen, which can be expressedin a temperature-sensitive fashion. The cells are stimulated to divideby being cultured at the temperature at which the antigen is expressed.Once the cells have formed a monolayer, they are placed at a temperatureat which the antigen is not expressed, causing the cells to stopdividing and differentiate. Differentiation results in the formation oftight junctions and the polarization of the plasma membranes. Monolayersof polarized cells are tested for the ability to transport agents,conjugates or conjugate moieties.

In some methods, the ability of an agent, conjugate, or conjugate moietyto specifically bind to the BGT1 transporter is tested. A knownsubstrate of the BGT1 transporter and the agent, conjugate, or conjugatemoiety are added to cells expressing the BGT1 transporter. The amount orrate of transport of the substrate in the presence of the agent,conjugate, or conjugate moiety is compared to the amount or rate oftransport of the agent, conjugate, or conjugate moiety in the absence ofthe test compound. If the amount or rate of transport of the substrateis decreased by the presence of the agent, conjugate, or conjugatemoiety, the agent, conjugate, or conjugate moiety binds the BGT1transporter. Agents, conjugates or conjugate moieties that bind the BGT1transporter can be further analyzed to determine if they are transportedby the BGT1 transporter or only adhere to the exterior of thetransporter. Agents, conjugates or conjugate moieties that aretransported by the BGT I transporter can be further tested to determineif they are transported from one side of a monolayer of polarized cellsto the other side, such as a monolayer of brain microvessel endothelialcells. Agents and conjugates having neuropharmaceutical activity andthat are transported by the BGT1 transporter can be used to formpharmaceutical compositions. Conjugate moieties that are transported bythe BGT1 transporter can be linked to a therapeutic or cytotoxicneuropharmaceutical agent or an imaging component.

Transport of a compound into a cell can be detected by detecting asignal from within a cell from any of a variety of reporters. Thereporter can be as simple as a label such as a fluorophore, achromophore, or a radioisotope. Confocal imaging can also be used todetect internalization of a label as it provides sufficient spatialresolution to distinguish between fluorescence on a cell surface andfluorescence within a cell; alternatively, confocal imaging can be usedto track the movement of compounds over time. In another approach,transport of a compound is detected using a reporter that is a substratefor an enzyme expressed within a cell. Once the compound is transportedinto the cell, the substrate is metabolized by the enzyme and generatesan optical signal that can be detected. Light emission can be monitoredby commercial PMT-based instruments or by CCD-based imaging systems. Inaddition, assay methods utilizing liquid chromatography-massspectroscopy (LC-MS-MS) detection of the transported compounds orelectrophysiological signals indicative of transport activity are alsoemployed. Mass spectroscopy is a powerful tool because it allowsdetection of very low concentrations of almost any compound, especiallymolecules for which a radiolabeled version is not available. It can alsobe used to distinguish substrates from non-transported ligands. Thesesame detection methods can be used to determine if a compound istransported from one side of a monolayer of polarized cells to the otherside by administering the compound to one side of the monolayer andsampling the media on the other side of the monolayer after apredetermined period of time.

In some methods, multiple agents, conjugates or conjugate moieties arescreened simultaneously and the identity of each agent, conjugate, orconjugate moiety is tracked using tags linked to the agents, conjugatesor conjugate moieties. In some methods, a preliminary step is performedto determine binding of an agent, conjugate, or conjugate moiety to atransporter. Although not all agents, conjugates or conjugate moietiesthat bind to a transporter are substrates of the transporter,observation of binding is an indication that allows one to reduce thenumber of candidates from an initial repertoire. In some methods, thetransport rate of an agent, conjugate, or conjugate moiety is tested incomparison with the transport rate of a reference substrate for thattransporter. For example, GABA, a natural substrate of BGT1, can be usedas a reference. The comparison can be performed in separate parallelassays in which an agent, conjugate, or conjugate moiety under test andthe reference substrate are compared for uptake on separate samples ofthe same cells. Alternatively, the comparison can be performed in acompetition format in which an agent, conjugate, or conjugate moietyunder test and the reference substrate are applied to the same cells.Typically, the agent, conjugate, or conjugate moiety and the referencesubstrate are differentially labeled in such assays.

In comparative assays, the V_(max) of an agent, conjugate, or conjugatemoiety tested can be compared with that of a reference substrate. If anagent, conjugate moiety, or conjugate has a V_(max) of at least 1%, 5%,10%, 20%, and most preferably at least 50% of the reference substratefor the BGT1 transporter, then the agent, conjugate moiety, or conjugateis also a substrate for the BGT1 transporter. If transport of the agent,conjugate moiety, or conjugate into the CNS is desired, a higher V_(max)of the agent, conjugate moiety, or conjugate relative to that of thereference substrate is preferred. Therefore, agents, conjugate moieties,or conjugates having V_(max)'s of at least 1%, 5%, 10%, 20%, 50%, 100%,150%, or 200% (i.e., two-fold) of the V_(max) of a reference substrate(e.g., GABA) for the transporter are screened in some methods. Thecomponents to which conjugate moieties are linked can by themselves showlittle or no detectable substrate activity for the transporter (e.g.,V_(max) relative to that of a reference substrate of less than 0.1% or1%). Preferred agents, conjugates, or conjugate moieties have a V_(max)for BGT1 that is at least 5% of the V_(max) for BGT1 of GABA. Preferredconjugates comprising a neuropharmaceutical agent or imaging componentlinked to a conjugate moiety preferably have a greater V_(max) for BGT1than the neuropharmaceutical agent or imaging component alone.

Having determined that an agent, conjugate, or conjugate moiety is asubstrate for BGT1, a further screen can be performed to determine itstherapeutic activity in treatment or prophylaxis of a disease, or itscytotoxic activity against brain tumor cells. Usually the disease isneurological (i.e., the pathology occurs in the CNS). Alternatively, thediseased tissue is non-CNS tissue but is responsive to treatment by anagent that exerts a pharmacological effect on the CNS that in turncauses an effect on the diseased non-CNS tissue, such as an effectcaused by the release of hormones from the CNS. Diseases of this typeare also considered to be diseases of the CNS unless otherwise apparentfrom context. If the agent, conjugate, or conjugate moiety does not haveinherent therapeutic or cytotoxic activity, it is first linked toanother chemical component having such therapeutic or cytotoxicproperties. The agent, conjugate, or conjugate moiety is then contactedwith cells expressing BGT1. The contacting can be performed either on apopulation of cells in vitro, or the brain microvessel endothelial cellsof a test animal via administration of the agent, conjugate, orconjugate moiety to a test animal. The therapeutic or cytotoxic activityof the agent, conjugate, or conjugate moiety is then determined fromestablished protocols for that particular disease. Optionally, theeffect of the agent, conjugate, or conjugate moiety can be compared witha placebo.

A further screen can be performed to determine toxicity of the agent,conjugate, or conjugate moiety to normal cells. The agent, conjugate, orconjugate moiety is administered to a laboratory animal that ispreferably in an un-diseased state. Various tissues of the animal, suchas liver, kidney, heart, and brain are then examined for signs ofpathology. Cells in the animal can also be analyzed for uptake of theagent, conjugate, or conjugate moiety.

IV. Iterative Modification and Testing of BGT1 Substrates

Having determined that an agent, conjugate, or conjugate moiety is asubstrate for BGT1, the agent, conjugate, or conjugate moiety can bemodified to improve its properties as a substrate. The modified agent,conjugate, or conjugate moiety is then tested for transport by BGT1.Modified agents, conjugates, or conjugate moieties that are transportedby BGT1 at a higher V_(max) compared to the unmodified agents,conjugates, or conjugate moieties are preferred. The process ofmodifying agents, conjugates, or conjugate moieties and testing fortransport by BGT1 can be repeated until a desired level of transport isreached.

Agents, conjugates or conjugate moieties that are substrates of BGT1 canalso be modified for decreased capacity to be transported out of cellsby efflux transporters. An agent, conjugate, or conjugate moietytransported by BGT1 is assayed to determine whether it is also asubstrate for one or more efflux transporters. If the agent, conjugate,or conjugate moiety is transported by an efflux transporter, the agent,conjugate, or conjugate moiety is modified and tested for both reducedtransport by an efflux transporter and retention of BGT1 substrateactivity.

In some instances, the specific efflux transporter responsible fortransporting an agent, conjugate, or conjugate moiety is known. Theagent, conjugate, or conjugate moiety is modified, preferably byaddition of a chemical group that differs in chemical characteristicsfrom other known substrates of the efflux transporter. The modifiedagent, conjugate, or conjugate moiety is then tested for retainedcapacity to be transported by BGT1 and a diminished capacity to betransported by an efflux transporter. It is not necessary that themodified agent, conjugate, or conjugate moiety retain the same kineticproperties of BGT1 transporter substrate as the unmodified agent,conjugate, or conjugate moiety as long as some BGT1 substrate activityis retained. Examples of efflux transporters are the P-glycoprotein(PgP), multidrug resistance protein (MRP1), and breast cancer resistanceprotein (BCRP). Preferred agents, conjugates, or conjugate moieties havea BGT1 transport:efflux transport ratio of at least 1.1:1.0, morepreferably, 2.0:1.0, and more preferably 5.0:1.0 and more preferably10.0:1.0 or higher at a given concentration of agent, conjugate, orconjugate moiety.

Efflux transporter activity can be measured in several ways. First,functional assays can be performed in which interaction of compoundswith efflux transporters is measured by stimulation of effluxtransporter ATPase activity in cellular membrane fragments or vesicles.Second, competition assays can be performed in which test compoundscompete with known efflux substrates in whole cells. Third, directtransport assays can be performed in which the transport of compounds ismeasured across a polarized monolayer of cells. Other assays besidesthese three can also be used to directly or indirectly measure theefflux substrate characteristics of a test compound.

The efflux transporter ATPase assay is based on the fact that mostefflux substrates increase the ATPase activity of efflux transportersupon binding. In one type of assay, Baculovirus membrane fragments orvesicles containing an efflux transporter such as PgP, as well ascontrol membrane fragments or vesicles not containing the effluxtransporter, are either prepared or obtained from commercial suppliers.The ATPase activity of the membrane fragments or vesicles is measured inthe presence of various concentrations of the test compound. An agent,conjugate, or conjugate moiety that is transported by BGT1 is added tothe ATPase assay reaction and the amount of ATPase activity is measuredat various concentrations of agent, conjugate, or conjugate moiety.Parallel experiments are performed in which ATPase activity is measuredunder addition of the same concentrations of modified agent, conjugate,or conjugate moiety that retain BGT1 substrate activity. Reduced ATPaseactivity caused by the modified agent, conjugate, or conjugate moietycompared to the unmodified agent, conjugate, or conjugate moietyindicates that the modified agent, conjugate, or conjugate moiety is abetter candidate for retention in the CNS.

In the competition assay, the test compound is assayed for competitionwith a known efflux substrate. For example, calcein-AM is anon-fluorescent compound that is a substrate of PgP and MRP1. Calcein-AMis initially loaded into the cells, for example, by transport by passivediffusion. Cells expressing these efflux transporters actively effluxnearly all of the calcein-AM that is present in the cells. However, whenother efflux transporter substrates are present, these other substratescompete with calcein-AM for efflux, resulting in more calcein-AMaccumulating inside the cells. Intracellular esterases convert thenon-fluorescent calcein-AM to fluorescent calcein, which can be measuredspectrophotometrically. An agent, conjugate, or conjugate moiety that istransported by BGT1 is loaded into efflux transporter-containing cellsby either BGT1 transport or passive diffusion. Calcein-AM is also loadedinto the cells by active transport or transport by passive diffusion.Accumulation of calcein-AM is measured and compared to the amount ofaccumulation in the absence of the agent, conjugate, or conjugatemoiety. Parallel experiments are performed in which a modified agent,conjugate, or conjugate moiety that is transported by BGT1 is loadedinto the cells. Accumulation of calcein-AM is measured and compared tothe amount of accumulation in the absence of the modified agent,conjugate, or conjugate moiety. Decreased calcein-AM accumulation insidethe cells caused by the presence of a modified agent, conjugate, orconjugate moiety compared to calcein-AM accumulation in the presence ofunmodified agent, conjugate, or conjugate moiety indicates that themodified agent, conjugate, or conjugate moiety is a better candidate forretention inside the CNS.

The cells used for competition assays can be cells that either express ahigh endogenous level of the efflux transporter of interest or aretransformed with an expression vector containing the efflux transportergene. Suitable cell lines for efflux assays are, for example, HEK andMDCK cell lines into which the PgP gene has been transfected, orMES-SA/Dx5 uterine sarcoma cells grown in the presence of 500 nMdoxorubicin, which express a high endogenous level of PgP. These cellscan optionally be transfected with the BGT1 transporter gene. Preferredcells express one or more efflux transporter genes such as PgP and theBGT1 gene, either endogenously or through transfection of expressionvectors.

A third type of efflux transporter assay is the cellular transwellmonolayer efflux assay. In this assay, cells expressing effluxtransporters, such as MDCK, HEK, CHO, and LLCPK cells containing theTREx-PgP expression vector (Invitrogen Inc., Carlsbad, Calif.), areseeded and grown in transwell dishes on filter membranes made ofsubstances such as polycarbonate. The cells form a polarized monolayer.The transwell dishes have apical and basolateral chambers that areseparated by the filter membrane on which the polarized monolayer issituated. Assays are performed by placing a test compound in either theapical or basolateral chamber, followed by sampling the opposite chamberafter a predetermined period of time such as 60-120 minutes andmeasuring the amount of the test compound. The test compound can bemeasured by methods such as radiolabel detection or LC-MS-MS analysis.Assays are performed in the presence and absence of an effluxtransporter inhibitor or competitor. Efflux transporter inhibitors orcompetitors increase apical to basolateral transport and decreasebasolateral to apical transport of compounds that are efflux transportersubstrates. Apparent permeability (P_(app)) of test compounds ismeasured. Test compounds that are substrates of efflux transportersgenerate a P_(app) (basolateral to apical)/P_(app) (apical tobasolateral) ratio of greater than 2.0, while test compounds that arenot substrates generate a ratio of 1.5 or less. Test compounds thatgenerate ratios between 1.5 and 2.0 require additional testing todetermine if they are efflux transporter substrates. An agent,conjugate, or conjugate moiety that is a BGT1 substrate and alsogenerates a ratio of greater than 2.0 can be modified. A modified agent,conjugate, or conjugate moiety that retains BGT1 substrate activity andgenerates a lower ratio compared to the unmodified agent, conjugate, orconjugate moiety indicates that the modified agent, conjugate, orconjugate moiety is a better candidate for retention inside the CNS.

An additional screen can be performed to determine whether agents,conjugates, or conjugate moieties have substantial capacity for passivediffusion across the brain microvessel endothelial cells making up theblood brain barrier. Such an assay can be performed using cells lackingBGT1 transporters. That is, the agents, conjugates, or conjugatemoieties are exposed to cells that lack BGT1 transporters, and theamount of agents, conjugates, or conjugate moieties that are presentinside the cell is measured.

V. Modification of Compounds Having Non-Neuropharmacologic Activity

In some instances it is desirable to modify an agent to reduce itscapacity to be transported from the blood into the brain. Reducedcapacity to enter the brain is desirable for agents having apharmacological activity that is useful in a tissue outside the CNS, butwhich causes undesired side effects when the agent enters the CNS. Mosttypically, such agents are drugs administered to treat anon-neurological disease, and which exert a useful therapeuticpharmacological effect on cells, tissues, or molecules located outsideof the CNS. When such drugs are transported from the blood into thebrain, serious side effects can occur. Many known drugs exhibitundesirable side effects from penetrating the CNS. Examples includedrowsiness experienced by patients taking antihistamines, nonsteroidalanti-inflammatory drugs (NSAIDS), anti-asthmatics, andantihypertensives.

Some methods are performed on an agent having an intended site ofpharmacological activity that is located outside of the CNS. The agentcan be known or suspected to enter the CNS. In some instances, the agentis known to be transported by BGT1. The agent is covalently attached toa conjugate moiety and the resulting conjugate is tested for transportinto the brain. The assay can be performed on brain microvesselendothelial cells, cells transformed with a BGT1 expression vector, apolarized monolayer of cells, or an actual blood brain barrier viaadministration to a test animal. Transport of the conjugate is thencompared with transport of the agent alone (i.e., without the conjugatemoiety). Conjugates having a lower V_(max) for transport than the agentalone are less likely to exhibit undesirable CNS side effects caused byunwanted transport from the blood into the brain. For example, preferredconjugates include those having a lower V_(max) for transport by BGT1than the agent alone.

Some methods comprise providing an agent having a pharmacologicalactivity, wherein the pharmacological activity is useful for treating adisease present in a tissue other than the CNS, and the pharmacologicalactivity results in undesired side effects in the CNS if the agententers the CNS, modifying the agent, providing a cell expressing atleast one transporter protein that transports substrates across theblood brain barrier, contacting the cell with the modified agent, anddetermining whether the modified agent passes through the plasmamembrane via the transporter protein with a lower V_(max) than theagent, a lower V_(max) indicating that the modification decreases thecapacity of the modified agent relative to the agent to cross the bloodbrain barrier, thereby decreasing undesired side effects in the CNS. Insome methods the at least one transporter protein is BGT1. In somemethods the cell is transformed or injected with a nucleic acid encodinga transporter or the cell is a brain microvessel endothelial cell. Insome methods the modifying step comprises linking the agent to aconjugate moiety to form a conjugate, preferably wherein the conjugatemoiety is an inhibitor of the BGT1 transporter.

Other methods comprise providing an agent having a pharmacologicalactivity, wherein the pharmacological activity is useful for treating adisease present in a tissue other than the CNS, and the pharmacologicalactivity results in undesired side effects in the CNS if the agententers the CNS, modifying the agent, providing a cell expressing atleast one efflux transporter protein that transports substrates out ofthe CNS, contacting the cell with the modified agent, and determiningwhether the modified agent is transported by the at least one effluxtransporter protein with a higher V_(max) than the agent, a higherV_(max) indicating that the modification increases the capacity of themodified agent relative to the agent to be transported out of the CNS,thereby decreasing undesired side effects in the CNS. In some methodsthe at least one efflux transporter protein is P-glycoprotein (PgP),multidrug resistance protein (MRP1), or breast cancer resistance protein(BCRP). In some methods the cell is transformed or injected with anucleic acid encoding an efflux transporter or the cell is a brainmicrovessel endothelial cell, a kidney-derived cell, or a uterinesarcoma cell. In some methods the modifying step comprises linking theagent to a conjugate moiety to form a conjugate, preferably wherein theconjugate moiety is a substrate of the efflux transporter.

VI. Sources of Neuropharmaceutical Agents, Imaging Components, andConjugate Moieties

Therapeutic neuropharmaceutical agents, cytotoxic neuropharmaceuticalagents, imaging components and conjugate moieties can be obtained fromnatural sources such as, e.g., marine microorganisms, algae, plants, andfungi. Alternatively, these compounds can be from combinatoriallibraries, including peptides or small molecules, or from existingrepertories of chemical compounds synthesized in industry, e.g., by thechemical, pharmaceutical, environmental, agricultural, marine,cosmeceutical, drug, and biotechnological industries.Neuropharmaceutical compounds can include proteins, antibiotics,adrenergic agents, anticonvulsants, small molecules, nucleotide analogs,chemotherapeutic agents, anti-trauma agents, peptides and other classesof agents used in treatment or prophylaxis of a neurological disease.Examples of such proteins include CD4 (including soluble portionsthereof), growth factors (e.g., nerve growth factor and interferon),dopamine decarboxylase and tricosanthin. Examples of such antibioticsinclude amphotericin B, gentamycin sulfate, and pyrimethamine. Examplesof such adrenergic agents (including blockers) include dopamine andatenolol. Examples of such chemotherapeutic agents include adriamycin,methotrexate, cyclophosphamide, etoposide, and carboplatin. An exampleof an anticonvulsant that can be used is valproate and an anti-traumaagent that can be used is superoxide dismutase. Examples of suchpeptides are somatostatin analogues and enkephalinase inhibitors.Nucleotide analogs that can be used include azido thymidine (hereinafterAZT), dideoxy Inosine (ddI), and dideoxy cytodine (ddc).

Typically if an agent is being screened as a substrate, the agent isknown or suspected to have an inherent therapeutic neuropharmaceutical,cytotoxic neuropharmaceutical or imaging activity. If a conjugate isbeing screened, the conjugate usually comprises such an agent orcomponent. If a conjugate moiety is being screened, the conjugate moietytypically lacks a therapeutic, cytotoxic, or imaging activity and anagent or component that has this activity is added after screening.

Suitable cytotoxic agents for incorporation into conjugates or linkageto conjugate moieties after screening include platinum, nitrosourea,nitrogen mustard, nitroimidazole, and a phosphoramide group that is onlycytotoxic to brain tumor cells. The choice of imaging component dependson the means of detection. For example, a fluorescent imaging componentis suitable for optical detection. A paramagnetic imaging component issuitable for topographic detection without surgical intervention.Radioactive labels can also be detected using positron emissiontomography or single photon emission computed tomography.

The agents, conjugates or conjugate moieties to be screened, optionallylinked to a neuropharmaceutical agent or an imaging component if notinherently present, are preferably small molecules having molecularweights of less than about 2000 Da, preferably less than about 1500 Da,preferably less than about 1000 Da and preferably less than about 500Da.

VII. Linkage of Neuropharmaceutical Agents or Imaging Components toSubstrates

Conjugates can be prepared either by direct conjugation of aneuropharmaceutical agent or an imaging component to a substrate of BGT1with a covalent bond (optionally cleavable in vivo), or by covalentlycoupling a difunctionalized linker precursor with theneuropharmaceutical agent or imaging component and substrate. The linkerprecursor is selected to contain at least one reactive functionalitythat is complementary to at least one reactive functionality on theneuropharmaceutical agent or imaging component and at least one reactivefunctionality on the substrate. Optionally, the linker is cleavable.Suitable complementary reactive groups are well known in the art asillustrated below:

Complementary Binding Chemistries

First Reactive Group Second Reactive Group Linkage hydroxyl carboxylicacid ester hydroxyl haloformate carbonate thiol carboxylic acidthioester thiol haloformate thiocarbonate amine carboxylic acid amidehydroxyl isocyanate carbamate amine haloformate carbamate amineisocyanate urea carboxylic acid carboxylic acid anhydride hydroxylphosphorus acid phosphonate or phosphate ester

The same methods of chemical modification can be used to form conjugatesfor the purpose of inhibiting transport into the CNS, for inhibitingefflux from the CNS, or for enhancing efflux from the CNS.

VIII. Pharmaceutical compositions

The above screening processes can identify one or more types ofcompounds that can be incorporated into pharmaceutical compositions.These compounds include agents that are both substrates for BGT1 andhave an inherent neuropharmaceutical activity or imaging activity. Thecompounds also include conjugates in which a neuropharmaceutical agentor imaging component is linked to a substrate for BGT1. Conjugatescomprising an agent with a pharmacological activity and a conjugatemoiety having decreased substrate capacity for BGT1 relative to theagent alone are also provided for the purpose of reducing transport ofthe agent into the CNS, where the agent would confer undesired sideeffects.

One or more of the above entities can be combined withpharmaceutically-acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, buffered water, physiologicalsaline, phosphate buffered saline (PBS), Ringer's solution, dextrosesolution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation can also include other carriers, adjuvants,or non-toxic, nontherapeutic, nonimmunogenic stabilizers, excipients,and the like. The compositions can also include additional substances toapproximate physiological conditions, such as pH adjusting and bufferingagents, toxicity adjusting agents, wetting agents, detergents, and thelike (see, e.g., Remington's Pharmaceutical Sciences, Mace PublishingCompany, Philadelphia, Pa., 17th ed. (1985); for a brief review ofmethods for drug delivery, see, Langer, Science 249:1527-1533 (1990);each of these references is incorporated by reference in its entirety).

Pharmaceutical compositions can be administered orally, intranasally,intradermally, subcutaneously, intrathecally, intramuscularly,topically, intravenously, or injected directly to a site of canceroustissue. For parenteral administration, the compounds disclosed hereincan be administered as injectable dosages of a solution or suspension ofthe compound in a physiologically acceptable diluent with apharmaceutical carrier which can be a sterile liquid such as water,oils, saline, glycerol, or ethanol. Additionally, auxiliary substances,such as wetting or emulsifying agents, surfactants, pH bufferingsubstances, and the like can be present in the pharmaceuticalcompositions. Other components of pharmaceutical compositions are thoseof petroleum, animal, vegetable, or synthetic origin, for example,peanut oil, soybean oil, and mineral oil. In general, glycols such aspropylene glycol or polyethylene glycol are preferred liquid carriers,particularly for injectable solutions.

Typically, compositions are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid vehicles prior to injection can also be prepared.The preparation also can be emulsified or encapsulated in liposomes ormicro particles such as polylactide, polyglycolide, or a copolymerthereof for enhanced adjuvant effect, as discussed above (see Langer,Science 249, 1527 (1990) and Hanes, Advanced Drug Delivery Reviews 28,97-119 (1997)). The pharmaceutical compositions disclosed herein can beadministered in the form of a depot injection or implant preparationthat can be formulated in such a manner as to permit a sustained orpulsatile release of the active ingredient.

Pharmaceutical compositions for oral administration can be in the formof e.g., tablets, pills, powders, lozenges, sachets, cachets, elixirs,suspensions, emulsions, solutions, or syrups. Some examples of suitableexcipients include lactose, dextrose, sucrose, sorbitol, mannitol,starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin,calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,cellulose, sterile water, syrup, and methylcellulose. Preserving agentssuch as methyl- and propylhydroxy-benzoates; sweetening agents; andflavoring agents can also be included. Depending on the formulation,compositions can provide quick, sustained, or delayed release of theactive ingredient after administration to the patient. Polymericmaterials can be used for oral sustained release delivery (see “MedicalApplications of Controlled Release,” Langer and Wise (eds.), CRC Pres.,Boca Raton, Fla. (1974); “Controlled Drug Bioavailability,” Drug ProductDesign and Performance, Smolen and Ball (eds.), Wiley, N.Y. (1984);Ranger and Peppas, 1983, J Macromol. Sci. Rev. Macromol Chem. 23:61; seealso Levy et al., 1985, Science 228: 190; During et al., 1989, Ann.Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). Sustainedrelease can be achieved by encapsulating conjugates within a capsule, orwithin slow-dissolving polymers. Preferred polymers include sodiumcarboxymethylcellulose, hydroxypropylcellulose,hydroxypropylmethylcellulose, and hydroxyethylcellulose (most preferred,hydroxypropyl methylcellulose). Other preferred cellulose ethers havebeen described (Alderman, Int. J. Pharm. Tech. & Prod. Mfr., 1984, 5(3),1-9). Factors affecting drug release have been described in the art(Bamba et al., Int. J. Pharm., 1979, 2, 307). For administration byinhalation, the compounds for use according to the disclosures hereinare conveniently delivered in the form of an aerosol spray preparationfrom pressurized packs or a nebulizer, with the use of a suitablepropellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas, or frompropellant-free, dry-powder inhalers. In the case of a pressurizedaerosol the dosage unit can be determined by providing a valve todeliver a metered amount. Capsules and cartridges of, e.g., gelatin foruse in an inhaler or insufflator, can be formulated containing a powdermix of the compound and a suitable powder base such as lactose orstarch.

Effective dosage amounts and regimes (amount and frequency ofadministration) of the pharmaceutical compositions are readilydetermined according to any one of several well-established protocols.For example, animal studies (e.g., mice, rats) are commonly used todetermine the maximal tolerable dose of the bioactive agent per kilogramof weight. In general, at least one of the animal species tested ismammalian. The results from the animal studies can be extrapolated todetermine doses for use in other species, such as humans for example.

The components of pharmaceutical compositions are preferably of highpurity and are substantially free of potentially harmful contaminants(e.g., at least National Food (NF) grade, generally at least analyticalgrade, and more typically at least pharmaceutical grade).

To the extent that a given compound must be synthesized prior to use,the resulting product is typically substantially free of any potentiallytoxic agents, particularly any endotoxins, which may be present duringthe synthesis or purification process. Compositions are usually madeunder GMP conditions. Compositions for parenteral administration areusually sterile and substantially isotonic.

IX. Methods of Treatments

Pharmaceutical compositions disclosed herein are used in methods oftreatment of prophylaxis of neurological diseases. Examples of suchdiseases amenable to treatment are cancer (e.g., brain tumors), AcquiredImmune Deficiency Syndrome (AIDS), stroke, epilepsy, Parkinson'sdisease, multiple sclerosis, neurodegenerative disease, trauma,depression, Alzheimer's disease, migraine, pain, seizure disorders,inflammation, and allergic diseases.

Other pharmaceutical compositions disclosed herein are used in methodsof treatment and prophylaxis of non-neurological diseases. Examples ofsuch diseases amenable to treatment are cancer (e.g., tumors of non-CNStissue), inflammation, and allergic diseases.

In prophylactic applications, pharmaceutical compositions areadministered to a patient susceptible to, or otherwise at risk of, adisease in an amount and frequency sufficient to eliminate or reduce therisk, lessen the severity, or delay the outset of the disease, includingbiochemical, histologic, and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. In therapeutic applications, pharmaceuticalcompositions are administered to a patient suspected of, or alreadysuffering from such a disease in an amount and frequency sufficient tocure, or at least partially arrest, the symptoms of the disease(biochemical, histologic, and/or behavioral), including itscomplications and intermediate pathological phenotypes in development ofthe disease. An amount of pharmaceutical composition sufficient toachieve at least one of the above objects is referred to as an effectiveamount, and a combination of amount and frequency sufficient to achieveat least one of the above objects is referred to as an effective regime.

X. Methods of Imaging

As discussed above, the invention provides conjugates comprising aconjugate moiety, which are substrates of BGT1, linked to an imagingcomponent, as well as agents that are substrates for BGT1 and have aninherent imaging activity. Optionally, the agents also have inherentaffinity for a particular antigen or cell type found in the CNS, or theconjugate is provided with an additional conjugate moiety having suchaffinity. The additional moiety is referred to as a targeting moiety.The targeting moiety can be an antibody or fragment thereof, or anyother molecule that specifically binds to a desired antigen or cell typewithin the brain. The invention further provides pharmaceuticalcompositions comprising all of these entities. These pharmaceuticalcompositions can be used for in vivo imaging. The compositions areadministered to a patient and preferentially taken up by central nervoussystem cells after being actively transported from the blood into thebrain by brain microvessel endothelial cells expressing BGT1 in thepatient. The imaging activity is then detected. In some methods, theimaging component is also a cytotoxic agent. For example manyradioisotopes are suitable for both imaging and tumor cytotoxicactivity. In such cases, methods of imaging and methods of treatment canbe combined. Currently used diagnostic imaging techniques includepositron emission tomography (PET), magnetic resonance imaging (MRI),and computed tomography (CT). Actively transported imaging componentsprovide information about, for example, the presence and/or size of abrain tumor. The cell assay methods provided herein can also be used toidentify imaging compounds for use outside the CNS, wherein such imagingagents exert undesirable side effect on the CNS.

As can be appreciated from the disclosure above, the present inventionhas a wide variety of applications. For example, the BGT1 transportercan be used to identify an agent or conjugate that is a substrate forthe transporter and that can cross the blood brain barrier and cantherefore treat the CNS. The BGT1 transporter also can be used toincrease the capacity of an agent to cross the blood brain barrier byidentifying a conjugate moiety that is a substrate for the BGT1transporter and linking the conjugate moiety to the agent. Accordingly,the following examples are offered by way of illustration, not by way oflimitation.

EXAMPLES Example 1 Quantitative PCR Detection of BGT1 Expression inBrain Endothelial Cells

Quantitative PCR was performed to analyze hBGT1 expression in humanbrain endothelial cells. Human brain tissue was obtained from epilepticfoci surgically removed from human patients. Human brain microvesselendothelial cells were isolated as follows. The brain tissue was washedin 70% ethanol, and placed in sterile phosphate buffered saline.Meninges and surface vessels were removed. Cortical gray matter wasminced, placed in preparation medium (1 g/L glucose, 25 mM HEPES, 100U/ml penicillin, 100 μg/ml streptomycin, 10 μg/ml DNAse I, 1 mg/mlcollagenase/dispase, in DMEM, adjusted to a pH of 7.4) and incubated for1 hour at 37° C. Samples were centrifuged for 10 minutes at 1000×g. Fat,cell debris, and myelin were discarded. The pellet was resuspended infresh preparation medium and incubated for an additional 3 hours at 37°C. in a shaking bath. Medium was filtered through a 230 μM nylon sievefollowed by a 150 μM nylon sieve. Microvessels were collected byretention on a 60 μM nylon sieve. Capillaries were washed withpreparation medium, and then pelleted for RNA isolation.

Total RNA was isolated from the brain endothelial cells using thestandard protocol for the RNEasy RNA Isolation Kit (Qiagen). Cells wereresuspended in RLT lysis buffer at 10 ml per 0.4 grams of cells. Lysateswere vortexed and run through a QiaShredder Column (Qiagen) prior to RNAisolation. Once isolated, the RNA was quantified, run on a 1% agarosegel to ensure integrity, and then stored at −80° C.

Prior to cDNA synthesis, total RNA was DNAse I treated to destroygenomic DNA contamination (Invitrogen DNAseI Kit). Twenty microliters ofoligo dT primed single-stranded cDNA was then synthesized from 1 μgtotal RNA (Invitrogen Thermoscript cDNA Synthesis Kit). The cDNA wastreated with RNAse H and stored at −20° C.

Quantitative PCR was performed in a 96-well format using the MJ ResearchDNA Engine Opticon. For each transporter, a pair of 26-baseoligonucleotide primers was used to amplify the specific transporter.Primers were designed to recognize the non-conserved 3′ ends of hBGT1transporter mRNA. The single stranded cDNA was used as a template for aPCR reaction containing human, mouse or rat primers and SYBR Greenmaster mix (Applied Biosystems). Fluorescent signal was read and graphedeach cycle. A CT value, or cycle threshold value, was determined foreach reaction. This value was defined as the point at which thefluorescent signal of the reaction exceeds background fluorescence.Background fluorescence was calculated as 20 standard deviations abovethe average signal from cycles 3 through 10. Transcript abundance wasnormalized to GAPDH transcript levels. Averaged results from human BGT1amplification experiments are shown below in Table 1. The units ofmeasurement are mRNA transcripts detected per PCR reaction.

TABLE 1 BGT1 mRNA Expression in Human Capillary Endothelial Cellstranscripts forward primer reverse primer HUMAN 34,037 aacgcaaacccctgaaaggcagggggttaa BGT1 cttcatggatg tgggaagacac (SEQ ID NO: 2) (SEQ ID NO:3)

The enrichment of hBGT1 transcripts in brain capillary endothelial cells(BMECs) relative to total brain transcripts was also determined byquantitative PCR as described above. Total RNA was isolated from wholebrain samples. hBGT1 transcript levels were normalized to GLUT1transcript levels. GLUT1 transcript levels were determined using thehuman GLUTI primers described in Table 3 below. Table 2 below shows theaverage hBGT1 transcript levels, normalized transcript levels, and ratioof hBGT1 transcripts in BMEC versus human brain cells.

TABLE 2 hBGT1 mRNA Expression in Human Brain Microvessel EndothelialCells Average BMEC % BMEC:Brain BMEC GLUT1 Ratio BGT1 34,037 4.2 19.2GLUT1 802,859 100 43.1

To confirm the purity of the brain endothelial cell RNA preparations,samples of RNA from each preparation were tested by quantitative PCR formRNA transcript levels of capillary (GLUT1) and neuronal (BNPI) cellmarkers. The quantitative PCR analysis was conducted as described above.The primers used are shown in Table 6 below. The results of the controlgene transcript quantification are shown in Table 7 below.

TABLE 3 Primers for Quantitative Analysis of Control Genes Gene forwardprimer reverse primer Human ggggcatgattggctcctt aggccgcagtacacaccgaGLUT1 ctctgtg tgatgaa (SEQ ID NO: 4) (SEQ ID NO: 5) Humancaccccccgctttccttta ctgctggtaggggagatgt BNPI tctccag gaagtgg (SEQ ID NO:6) (SEQ ID NO: 7)

TABLE 4 Control Gene mRNA Transcript Levels Control Gene Human TissueSource GLUT1 (Capillary marker) Capillaries 802859 Whole Brain 11120BNPI (Neuronal marker) Capillaries 2614 Whole Brain 222285

Example 2 Studies of Cloned BGT1 Transporters: Oocyte Expression

To assess transport function of a specific transporter protein, it ispreferable to clone the cDNA and express the protein in cells that havelow endogenous transport activity. Human BGT1 is cloned by PCR, fullysequenced, and subcloned into plasmids that can be used for expressionin mammalian cells or Xenopus oocytes. For expression in Xenopusoocytes, in vitro BGT1 cRNA is prepared and injected into defoliculatedoocytes.

Oocytes expressing BGT1 protein exhibit higher levels of ³H-GABA uptakethan noninjected controls. To measure directly the uptake of possiblesubstrates, an oocyte uptake assay is performed in which uptake ofcompounds is measured by mass spectroscopy. For example, uptake of GABAcan be measured. Oocytes injected with BGT1 cRNA are incubated at 16-18°C. until maximal transporter expression is reached. Oocytes from thesame batch, not injected with cRNA, can be used as a control. A 0.5 mMsolution of GABA is prepared in oocyte ringers (ND96) buffer (90 mMNaCl, 10 mM HemiNa HEPES, 2 mM KCl, 1 mM MgCI₂, 1.8 mM CaCl₂, pHadjusted to 7.4) containing 0.5% bovine serum albumin. The GABA is, forexample, administered to pools of 8 oocytes for a 20 minute duration.Following the incubation, the pools of oocytes are washed with 0.5% BSAND96 buffer and separated into subpools containing, for example, 4oocytes each. Subpools are homogenized in 150 μl of ice cold 80%MeOH/H₂O and lysed manually. Lysates are vortexed before beingcentrifuged at, for example, 13.2 krpm for 15 minutes. Approximately 110μl of lysate is removed from the tubes and placed in a 96-well plate andanalyzed for GABA concentration by LC-MS-MS.

Samples are analyzed by LC-MS-MS as follows. A specific method can bedeveloped for each test compound, and calibrated against a series ofdilutions of known compound concentrations spiked into cellular extract.Measurements are performed using, for example, an API 2000 LC-MS-MSspectrometer equipped with Agilent 1100 binary transporters and a CTCHTS-PAL autosampler. Analyte fragmentation peaks are integrated, forexample, using Analyst 1.2 quantitation software, and concentrations arecalculated using a calibration curve of signals produced by knownconcentrations of the compound.

Example 3 Studies of Cloned BGT1 Transporters: BGT1 Transport Currentsin Oocytes

The BGT1 protein couples transport of GABA to the sodium gradient byco-transporting 2 sodium ions for each substrate molecule. Thus, thereis a net flux of positive charge into the cells during BGT1 transport.This net charge movement is measured as current using two-electrodevoltage clamping in oocytes expressing BGT1. The membrane potential ofoocytes is held at 60 mV and current traces were acquired using PowerLabsoftware (ADInstruments). Full 7-concentration dose-responses wereperformed for the test compound. Current responses at the highestconcentration are normalized to the maximal GABA concentration (2 mM).Half-maximal concentrations are calculated using non-linear regressioncurve fitting software (Prism) with the Hill co-efficient fixed to 1. Toensure that currents were specific for the over-expressed transporter,all compounds are tested against uninjected oocytes. Since BGT1 requiresNa⁺ for transport, transport specificity is confirmed by application ofthe test compounds in a Na⁺-free solution.

Example 4 Competition Assays

To determine whether a compound interacts with the BGT1 transporter, acompetition-binding assay is performed. This assay measures howdifferent concentrations of a test compound block the uptake of aradiolabeled substrate such as GABA. The half-maximal inhibitoryconcentration (IC₅₀) for inhibition of transport of a substrate by atest compound is an indication of the affinity of the test compound forthe BGT1 transporter. Competition binding studies are performed asfollows. Cells endogenously expressing the BGT1 transporter ortransfected with a BGT1 expression vector are plated in 96-well platesat 100,000 cells/well and incubated at 37° C. for 24 hours. RadiolabeledGABA (˜50,000 cpm/well) is added to each well in the presence andabsence of various concentrations of unlabeled GABA (γ-aminobutyricacid) in duplicate or triplicate. Plates are incubated at roomtemperature for 2 minutes. Excess radiolabeled GABA is removed and cellsare washed with cold assay buffer. Scintillation fluid is added to eachwell, and the plates are sealed and counted in a 96-well plate-basedscintillation counter. Data can be graphed and analyzed using non-linearregression analysis with Prism Software (GraphPad, Inc., San Diego,Calif.

Competition binding studies only demonstrate that a molecule interactswith the BGT1 protein, but do not demonstrate whether the molecule is asubstrate and is translocated across the plasma membrane, or is anon-transported inhibitor or a non-transported ligand. In order tomeasure whether test compounds are actively translocated across themembrane, and to determine the maximal transport rate, a direct uptakemethod is used in which transport of a test compound is measured by massspectroscopy. For direct uptake measurements using mass spectroscopy,cells are prepared similarly to those used for competition studies(described above). BGT1-expressing cells are washed and incubated withtest compounds such as GABA. Excess substrate is removed by washing withcold assay buffer. Cells are lysed with 50% ethanol/water and the celldebris is pelleted by centrifugation. The supernatant is analyzed byLC-MS-MS. As a negative control, uptake is measured in cells notexpressing BGT1 or by competition with another compound such as betaine.

Example 5 Efflux Assays

To determine whether a compound is a substrate for an effluxtransporter, an efflux assay is performed. The assay measures whether atest compound interacts with, or is a substrate for, the effluxtransporter.

Efflux assays can be performed by adding a test compound to commercialBaculovirus membranes (purchased from BD Biosciences) at variousconcentrations followed by ATPase activity measurement. A test compound(e.g., agent, conjugate, or conjugate moiety) that is a substrate of thetransporter of interest is added to the ATPase assay reaction and theamount of ATPase activity is measured at various concentrations of thetest compound. An efflux transporter substrate optionally is used as acontrol. Parallel experiments optionally can be performed in whichATPase activity is measured under the same conditions by addition of thesame concentrations of a modified test compound that retains transportersubstrate activity. Reduced ATPase activity caused by the modified testcompound compared to the unmodified test compound indicates that themodified compound is a better candidate for retention in the CNS.

The ATPase activity measurement is performed using the lactatedehydrogenase/pyruvate kinase coupled enzyme system described by Tietz &Ochoa, Arch. Biochim. Biophys. Acta 78:477 (1958) to follow the decreasein absorbance at 340 nm resulting from the oxidation of NADH, which isproportional to ATPase activity. 5 mM sodium azide (NaN₃), 1 mM EGTA,and 0.5 mM Ouabain, each of which inhibit non-specific ATPases in themembranes, are added to the reactions to further enhance the specificityof the PgP ATPase signal. The other components in the assay mixture are25 mM Tris, pH 7.8, 100 mM NaCl, 10 mM KCl, 5 mM MgCl₂, 1 mM DTT, 2 mMphosphoenolpyruvate, 1 mM NADH, 0.1 mg/ml lactate dehydrogenase, 0.1mg/ml pyruvate kinase, 5 mM ATP, and 6 μg PgP or control membranes. Forthe control, as the concentration of verapamil is increased, the ATPaseactivity in PgP-containing membranes, but not in control membraneswithout verapamil, also increased. Similarly, the ATPase activity inPgP-containing membranes, but not in control membranes, may increaseaccording to the binding or transport of the test compound by the effluxtransporter.

Efflux competition assays can be performed by transfecting atetracycline-inducible PgP expression construct (TREx-PgP) into HEKcells or other suitable cell line. The cell line optionally can betransfected with a nucleic acid encoding the transporter of interest.Cells are incubated with a PgP substrate, 5 μM calcein-AM, whichpassively diffuses into the cells, as well as with variousconcentrations of the test compound. Control cells are incubated with 5μM calcein-AM, as well as with various concentrations of the PgPsubstrate verapamil. As the concentration of PgP substrate verapamil isincreased, more calcein-AM accumulates in the cells and is converted tothe fluorescent product calcein. Similarly, if the test compound is aPgP substrate, calcein (converted from calcein-AM) will accumulate inthe cell. The accumulation of calcein is measured to determine thebinding or transport of the test compound (or a modified test compound)by the efflux transporter.

Transwell monolayer efflux assays can be performed by transfecting MDCKcells with the tetracycline-inducible TREx-PgP expression vector. Thecells optionally can be transfected with a nucleic acid encoding thetransporter of interest. The transfected cells are seeded onpolycarbonate filter membranes in transwell dishes and grown for 3-5days, yielding a polarized monolayer with tight junctions between cells.In this example, apical to basolateral and basolateral to apicaltransport of 2.5 nM (approximately 100,000 cpm) radiolabeled PgPsubstrate ³H-vinblastine is measured in the absence and presence of 250μM of the inhibitor/competitor verapamil. Apical to basolateraltransport of ³H-vinblastine is strongly increased and basolateral toapical transport of ³H-vinblastine is strongly decreased in the presenceof verapamil, indicating that ³H-vinblastine is a substrate of PgP. Fora test compound (or a modified test compound) the apical to basolateraltransport of, and basolateral to apical transport of, can be measuredand compared to determine the binding or transport of the test compoundby the efflux transporter.

Example 6 Recombinant BGT1 Expression

An inducible BGT1 expression construct was prepared. The human BGT1 cDNAwas linked to the tetracycline inducible promoter using the Gatewayplasmid cloning system following manufacturers instructions(Invitrogen). The tet-BGT1 expression construct was transfected intoHEK-TREx cells using Fugene transfection following manufacturer'sinstructions (Roche Biosciences). The resulting cell line was designateda HEK-TREx-BGT1 inducible cell line.

Example 7 hBGT1 Competition Uptake Assay

A modified competition uptake assay was developed to determine theability of a test compound(s) to inhibit the uptake of radiolabeledsubstrates into HEK-TREx-hBGT1 cells induced to over-express hBGT1. Theresults are stated as affinities (IC₅₀).

The competition uptake assay was prepared as follows: Compounds wereprepared for assay by diluting a 100 mM stock concentration (in DMSO) tothe appropriate working concentration. Typically, seven-point doseresponse curves were prepared starting at a final assay concentration of1 mM and carrying out three-fold dilutions. These dilutions wereprepared by making a working “compound” plate that contained a 2×solution of the desired starting concentration of each test compound induplicate in row A of a v-bottom microtiter plate. Six 3-fold serialdilutions (from row B to G) were made into the HBSS assay buffer (9.8g/L Hank's Balance Salts (Sigma; H-1387), 2.6 g/L HEPES (10 mM), (Sigma;H-3375), 0.35 g/L NaHCO₃ (4.2 mM) (Sigma; S-6297)), pH to 7.4 with 5NNaOH) with the appropriate amount of DMSO so that the DMSO concentrationremained constant at all dilutions. The resulting “compound” platecontained serial dilutions of six compounds in duplicate. The final row(H) of the assay plate was filled with HBSS buffer alone (H1-H6) or 10mM unlabeled GABA in HBSS (H7-H12) to measure the total or non-specificuptake, respectively.

³H-GABA was diluted into HBSS buffer to a final concentration of 4,000cpm/μl. Sufficient solution was prepared to allow addition of 25 μl/well(the final concentration was 100,000 cpm/well).

HEK-TREx-hBGT1 cells, plated in 96-well plates and treated with 2 mMbutyrate and tetracycline (or the tet analog, doxycycline), were removedfrom the CO₂ incubator. Growth media was removed from the cells, and thecells were washed twice in room temperature HBSS (100 μl/well/wash)using a 96-well plate washer (Bio Tek ELX405). Alternatively, cells werewashed manually with equivalent volumes using a multichannel pipettor.Immediately before beginning the assay, the final 100 μl wash solutionwas removed from the cells by aspiration.

Using a 96-well pipettor, 25 μl from the “compound” plate was added toeach well of the cell plate. The assay was started by adding 25 μl ofthe ³H-GABA working solution. The plate was incubated at roomtemperature for 5 minutes. The assay was stopped by washing the cellsfour times with ice-cold HBSS buffer using a ELX405 plate washer (100 μlbuffer/well/wash) having an angled buffer dispenser.

Scintillation fluid (200 μl) (Optiphase Supermix (Perkin Elmer) wasadded to each well, and the plate was covered with a 96-well adhesiveplate cover and placed on a shaker for 10 minutes. The plates werecounted on a 96-well plate scintillation counter for 60 sec/well. Thedata were analyzed using a sigmoidal dose response curve-fitting program(Prism, GraphPad, Inc, San Diego, Calif.; equation: one-sitecompetition).

Example 8 hBGT1 Direct Uptake Assay

A modified direct uptake assay was developed to determine the ability oftest compounds to be transported into HEK-TREx cells induced toover-express hBGT1. Four concentrations (bracketing the affinity asmeasured by competition assays) per compound were routinely tested.Non-specific uptake was determined by measuring the uptake into cellsnot induced to express the transporter (“no tet”).

The direct uptake assays were prepared as follows: Compounds wereprepared for assay by diluting a 100 mM stock concentration (in DMSO orwater, depending on compound solubility) to the appropriate workingconcentration. Typically, four concentrations bracketing the IC₅₀ weretested. The highest test concentration for each compound was made in anEppendorf tube and diluted into HBSS. The samples were robustly vortexedand centrifuged for 10 minutes at 13,200 rpm to spin down anyprecipitate. The supernatant from these samples (˜150 μl/well) wascarefully removed and placed into six wells of row A (cmpd 1: A1-6; cmpd2: A7-12) or row E (cmpd 3: E1-6; cmpd 4: E7-12) of a 96-wellpolypropylene “compound” plate. Three additional 2-fold dilutions weremade in the subsequent rows (B-D or F-G) in HBSS. With this set-up, fourcompounds were tested per plate: four concentrations of each compound intriplicate on cells that had either been induced (A: plus tet) or notinduced (B: no tet) to express the transporter.

An internal standard of 50 μM propranolol in 50:50 ethanol:water wasalso prepared. To prepare standard curves, several concentrations of thetest compounds were diluted into HEK cell extract (prepared fromtet-treated, mock-incubated, and extracted cells as described below)with a final internal standard concentration of 5 μM. Standards of 10,5, 1, 0.5, 0.1, 0.05, 0.01, and 0.005 μM were routinely run for eachtest compound.

HEK-TREx-hBGT1 cells were plated in 96-well plates and treated with 2 mMbutyrate and tetracycline (or the tetracycline analog, doxycycline;columns 1-3 and 7-9) or 2 mM butyrate without tetracycline (or thetetracycline analog, doxycycline; columns 4-6 and 10-12). The cells wereremoved from the CO₂ incubator, and the growth media was removed fromthe cells. The cells were washed twice in room temperature HBSS (100μl/well/wash) using a 96-well plate washer (Bio Tek ELX405).Alternatively, cells were washed manually with equivalent volumes usinga multichannel pipettor. Immediately before the assay was begun, thefinal 100 μl wash solution was removed from the cells. The assay wasstarted by using a 96-well pipettor to add 50 pl from the “compound”plate to each well of the HEK-TREx-hBGT1 cell plate. The plate wasincubated at room temperature for 5 minutes.

The assay was stopped by washing the cells four times with ice-cold HBSSbuffer using a ELX405 plate washer (100 μl buffer/well/wash) with anangled buffer dispenser. After the final wash, as much of the washbuffer as possible was removed by aspirating the wells with a probe thatreached the bottom of the wells. (Residual salts from the wash buffercan adversely affect the LC-MS-MS by disrupting the LC method or bysuppressing the MS signal.) 150 μl of a 50:50 ethanol:water solution wasadded to each well to lyse the cells and extract the test compound. Theplate was covered and allowed to sit for 20 minutes at room temperatureto ensure cell lysis. (The 50% ethanol solution is the generic solutionfor extraction. For compounds soluble in water or other solventscompatible with the LC-MS-MS, such solutions can be tried to determinewhether they interfere with the LC-MS-MS analysis. If the organicsolvent concentration is too high (>50%), it may be detrimental to theLC run).

120 μl of the cell extract was removed from each well and transferred toa fresh 96-well v-bottom polypropylene plate. This plate was coveredwith an adherent cover (top seal) and centrifuged for 15 minutes at5,700 rpm (Allegra 25R centrifuge) at 4° C. to pellet any cellprecipitate. 10 μl of the 50 μM propranolol solution was added to eachwell of a fresh 96-well plate (a plate amenable for sampling in theLC-MS-MS). 90 μl of the supernatant from the centrifuged cell extractwas removed and transferred to the plate containing the 10 μl ofpropranolol (using a Cybi-well 96-well pipettor). The sample plate witha bubble lid suitable for use with the LC-MS-MS and placed on a plateshaker for 5 to 10 minutes to mix the sample and the propranolol. Thesamples were submitted for LC-MS-MS analysis. The levels ofintracellular compounds were determined by converting the peak area toconcentration by extrapolating from the standard curve for eachcompound. Uptake was expressed as μM/well.

While the uptake values in these experiments were expressed as“μM/well,” the values can be easily converted to “pmol/well” or“pmol/well/unit time.” All of the samples were extracted with 150 μl ofextraction buffer. To convert the extrapolated “μM/well” values to“pmol/well,” the following equation can be used:X(10⁻⁶ mol/L)/well×150×10⁻⁶L=150×X pmol/well,where X=the extrapolated value obtained from the standard curve.

Example 9 GABA Competition Assay with HEK-TREx-BGT1 Cells

FIG. 2 depicts the results of a competition experiment usingHEK-TREx-BGT1 cells. ³H-GABA was used as the substrate and unlabeledGABA was used as the competitor. The competition experiment wasperformed as described in Example 7. Non-specific uptake was determinedby measuring the uptake into cells not induced to express thetransporter (“no tet”). FIG. 2 demonstrates that in cells induced toexpress BGT1, the uptake of labeled GABA decreased as the concentrationof unlabeled GABA increased. In control cells, uptake of labeled GABAremained at background levels and was largely unaffected by an excess ofunlabeled GABA.

Example 10 Competition and Direct Uptake Assays with HEK-TREx-hBGT1Cells Using GABA

GABA was used as the control substrate to characterize the competitionand direct uptake assays for hBGT1-expressing cells according to themethods described in Examples 7 and 8. A summary of the affinity andV_(max) values for GABA to hBGT1-expressing cells is provided in Table5.

FIG. 2 shows the results of the competition assay for ³H-GABA in thepresence of various concentrations of unlabeled GABA. FIG. 3 shows theresults of the direct uptake assay for GABA into HEK-TREx-hBGT1 cellsinduced to express hBGT1.

TABLE 5 Results of Competition and Direct Uptake Assays Direct UptakeCompetition Vmax BGT1 IC₅₀ (μM) K_(m) (μM) (pmol/min/well) GABA 20 27110

Although the foregoing compounds, conjugates, and methods have beendescribed in detail for purposes of clarity of understanding it will beobvious that certain modifications may be practiced within the scope ofthe claim(s) granted here from. Unless otherwise apparent from thecontext, any element, embodiment, or step can be used in combinationwith any other. All publications and patent documents cited herein arehereby incorporated by reference in their entirety for all purposes tothe same extent as if each were so individually denoted.

1. A method of screening a conjugate or conjugate moiety for capacity tobe transported through the blood brain barrier, comprising: (a)providing a cell expressing the BGT1 transporter, the BGT1 transporterbeing situated in the plasma membrane of the cell, wherein the BGT1transporter is the protein encoded by SEQ ID NO: 1 or has at least 90%sequence identity to the protein encoded by SEQ ID NO: 1, and the BGT1transporter can transport betaine; (b) contacting the cell with aconjugate or conjugate moiety; (c) determining that the conjugate orconjugate moiety passes through the plasma membrane via the BGT1transporter, passage through the BGT1 transporter indicating thecapacity to be transported through the blood brain barrier; (d) if step(b) comprises contacting the cell with a conjugate moiety and step (c)determines that the conjugate moiety passes through the plasma membranevia the BGT1 transporter, then linking the conjugate moiety to aneuropharmaceutical agent or to an imaging component to form a conjugateand repeating steps (a) through (c) with the conjugate; and (e)administering the conjugate or conjugate moiety to a peripheral tissueof an animal and measuring the amount of conjugate or conjugate moietythat passes through the blood brain barrier into the brain of theanimal; wherein the conjugate comprises a conjugate moiety linked to aneuropharmaceutical agent or to an imaging component.
 2. The method ofclaim 1, wherein: (i) the cell endogenously expresses the BGT1transporter; or (ii) a nucleic acid molecule encoding the BGT1transporter has been transfected or injected into the cell.
 3. Themethod of claim 2, wherein the cell is a brain microvessel endothelialcell.
 4. The method of claim 3, wherein the brain microvesselendothelial cell is one of a plurality of human brain microvesselendothelial cells forming a polarized monolayer, the conjugate orconjugate moiety is contacted to one side of the polarized monolayer,and the determining comprises determining whether the conjugate orconjugate moiety is transported into the brain microvessel endothelialcells or to the opposite side of the polarized monolayer.
 5. The methodof claim 2, wherein the cell is an oocyte.
 6. The method of claim 2,wherein the cell is a human embryonic kidney (HEK) cell.
 7. The methodof claim 2, wherein the cell is transformed with an SV40large T antigenthat can be expressed in a temperature sensitive fashion.
 8. The methodof claim 2, wherein the determining is performed by a net chargemovement assay.
 9. The method of claim 2, wherein the determining isperformed by a direct uptake assay.
 10. The method of claim 2, whereinthe determining is performed by a competition assay.
 11. The method ofclaim 1, wherein the determining step determines that the conjugate orconjugate moiety passes through the plasma membrane via the BGT1transporter; and the method further comprises: modifying the conjugateor conjugate moiety; and determining if the modified conjugate ormodified conjugate moiety is transported with a higher V_(max) by theBGT1 transporter than the conjugate or conjugate moiety.
 12. The methodof claim 1, wherein the neuropharmaceutical agent is a cytotoxicneuropharmaceutical agent selected from the group consisting ofplatinum, nitrosourea, a phosphoramide group that is selectivelycytotoxic to brain tumor cells, nitroimidazole, and nitrogen mustard.13. The method of claim 1, wherein the conjugate or conjugate moietycomprises an amino acid.
 14. The method of claim 13, wherein the aminoacid is selected from betaine, beta-alanine, and GABA.
 15. The method ofclaim 1, further comprising administering the conjugate or conjugatemoiety to an un-diseased animal and determining any toxic effects. 16.The method of claim 1, wherein the conjugate comprises a conjugatemoiety linked to a neuropharmaceutical agent.
 17. The method of claim 1,wherein the conjugate moiety is not a neuropharmaceutical agent or animaging component.
 18. The method of claim 1, wherein the conjugatecomprises a conjugate moiety cleavably linked to a neuropharmaceuticalagent or to an imaging component.
 19. The method of claim 1, wherein theconjugate comprises a conjugate moiety cleavably linked to aneuropharmaceutical agent and the conjugate does not havepharmacological activity.
 20. The method of claim 1, wherein the Vmax ofthe imaging component for the BGT1 transporter is less than 0.1% theVmax of GABA for the BGT1 transporter.
 21. The method of claim 1,wherein the conjugate comprises a conjugate moiety covalently coupled toa neuropharmaceutical agent or to an imaging component by adifunctionalized linker.
 22. The method of claim 1, wherein the linkingcomprises covalently coupling the conjugate moiety to theneuropharmaceutical agent or to the imaging component with adifunctional linking group.
 23. A method of screening a conjugate orconjugate moiety for capacity to be transported through the blood brainbarrier, comprising: (a) providing a cell expressing the BGT1transporter, the BGT1 transporter being situated in the plasma membraneof the cell, wherein the BGT 1 transporter is the protein encoded by SEQID NO: 1 or has at least 90% sequence identity to the protein encoded bySEQ ID NO: 1, and the BGT1 transporter can transport betaine; (b)contacting the cell expressing the BGT1 transporter with a conjugate orconjugate moiety; (c) determining that the conjugate or conjugate moietypasses through the plasma membrane via the BGT1 transporter, passagethrough the BGT1 transporter indicating the capacity to be transportedthrough the blood brain barrier; (d) if step (b) comprises contactingthe cell expressing the BGT1 transporter with a conjugate moiety andstep (c) determines that the conjugate moiety passes through the plasmamembrane via the BGT1 transporter, then linking the conjugate moiety toa neuropharmaceutical agent or to an imaging component to form aconjugate and repeating steps (a) through (c) with the conjugate; (e)providing a cell expressing at least one efflux transporter, wherein theat least one efflux transporter is selected from the glycoprotein (PgP)transporter, the multi-drug resistance protein (MRP1) transporter, andthe breast cancer resistance protein (BCRP) transporter; (f) contactingthe cell expressing at least one efflux transporter with the conjugateor conjugate moiety; and (g) determining that the conjugate or conjugatemoiety is transported by the at least one efflux transporter, anddetermining the efflux transporter activity of the conjugate orconjugate moiety; wherein the conjugate comprises a conjugate moietylinked to a neurophannaceutical agent or to an imaging component. 24.The method of claim 23, further comprising: determining the BGT1transporter activity of the conjugate or conjugate moiety; modifying theconjugate or conjugate moiety; establishing that the modified conjugateor conjugate moiety retains BGT1 transporter activity; determining theBGT1 transporter activity of the modified conjugate or modifiedconjugate moiety; determining the efflux transporter activity of themodified conjugate or modified conjugate moiety; and comparing the ratioof the BGT1 transporter activity to the ratio of efflux transporteractivity for the conjugate or conjugate moiety and the ratio of the BGT1transporter activity to the efflux transporter activity of the modifiedconjugate or modified conjugate moiety wherein an increased ratio ofBGT1 transporter activity to efflux transporter activity for themodified conjugate or modified conjugate moiety demonstrates that themodification improves the capacity of the conjugate or conjugate moietyto be transported through the blood brain barrier.
 25. The method ofclaim 23 or 24, wherein the efflux transporter activity is determined byconducting an assay selected from the group consisting of: (a) an effluxtransporter ATPase activity assay; (b) an efflux transporter competitionassay; and (c) a direct efflux transport assay across a polarizedmonolayer of cells.
 26. The method of claim 25, wherein the effluxtransporter activity is the V_(max) for the efflux transporter.
 27. Themethod of any one of claims 23 and 24, wherein the efflux transporteractivity is the V_(max) for the efflux transporter.
 28. A method ofscreening an agent or imaging component for decreased side effects inthe central nervous system (CNS), comprising: (a) providing (i) an agenthaving a pharmacological activity, wherein the pharmacological activityis useful for treating a disease present in a tissue other than the CNS,and the agent causes undesired side effects in the CNS if the agententers the CNS; or (ii) an imaging component useful for imaging a tissueother than the CNS, and the imaging component causes undesired sideeffects in the CNS if the imaging component enters the CNS; (b)modifying the agent or imaging component; (c) providing a cellexpressing the BGT1 transporter that transports substrates across theblood brain barrier, wherein the BGT1 transporter is the protein encodedby SEQ ID NO: 1 or has at least 90% sequence identity to the proteinencoded by SEQ ID NO: 1, and the BGT1 transporter can transport betaine;(d) contacting the cell with the agent or imaging component anddetermining that the agent or imaging component is a substrate of theBGT1 transporter; (e) determining the V_(max) of the agent or theimaging component for the BGT1 transporter; (f) contacting the cell withthe modified agent or modified imaging component and determining theV_(max) of the agent or imaging component for the BGT1 transporter; and(g) determining whether the modified agent or modified imaging componentpasses through the plasma membrane via the BGT1 transporter with a lowerV_(max) than the agent or imaging component, a lower V_(max) indicatingthat the modification decreases the capacity of the modified agent ormodified imaging component relative to the agent or imaging component tocross the blood brain barrier, thereby decreasing undesired side effectsin the CNS.
 29. The method of claim 28, wherein the cell is transformedor injected with a nucleic acid encoding a transporter or the cell is abrain microvessel endothelial cell.
 30. The method of claim 28, whereinthe modifying comprises linking the agent or imaging component to aconjugate moiety to form a conjugate.
 31. The method of claim 30,wherein the conjugate moiety is an inhibitor of the BGT1 transporter oris a substrate for an efflux transporter.
 32. The method of any one ofclaims 24 and 28, wherein the BGT1 transporter activity is the V_(max)for the BGT1 transporter.